This invention relates to antibodies having specificity for E-selectin characterised in that said antibodies are whole antibodies of neutral isotype, to processes for preparing said antibodies, to pharmaceutical compositions containing said antibodies, and to medical uses of said antibodies.
Selectins are a family of structurally related transmembrane glycoproteins implicated in the adhesion of leukocytes to vascular endothelial cells. The three known members, designated E-, P- and L- selectin are composed of three types of domain, an amino terminal C-type lectin domain, one EGF-like domain and between two and nine complementary regulatory repeats. Stimulation of endothelium by inflammatory cytokines e.g. II-1, or TNF results in the upregulation of E-selectin expression on the cell surface.
Experiments in vitro have shown that E-selectin can support the adhesion of polymorphonuclear cells, monocytes and a subpopulation of T-lymphocytes (see for example, Bevilacqua et al (1989) Science 243 1160-1165; Picker et al (1991) Nature 349 796-799 and Leeuwenberg et al (1992) Scant. J. Immunol 35 335-341). Mouse antibodies to E-selectin that block PMN binding in vitro have been shown to reduce extravasation of PMNs (neutrophils) in animal models (Mulligan, M. et al, J. Clinical Investigation 88 1396-1406 (1991) and Gundel, R. et al, J. Clinical Investigation 88 1407-1411 (1991)).
E-selectin thus appears to play a key role in the movement of leukocytes to sites of inflammation due to injury or infection. A corollary of this is that the expression of E-selectin is increased in certain inflammatory diseases. Hence E-selectin contributes to the disease process by supporting the adhesion of leukocytes which in turn cause tissue damage. It follows that an antibody to E-selectin that blocks this process would attenuate the extent or severity of the inflammation and hence be of therapeutic benefit.
Since most available monoclonal antibodies are of rodent origin, they are naturally antigenic in humans and thus can give rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response. Therefore, the use of rodent monoclonal antibodies as therapeutic agents in humans is inherently limited by the fact that the human subject will mount an immunological response to the antibody and will either remove it entirely or at least reduce its effectiveness.
Proposals have been made for making non-human MAbs less antigenic in humans using engineering techniques. These techniques generally involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule. A simple form of engineering antibodies involves the replacement of the constant regions of the murine antibody with those from a human antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81 6851-55; Whittle et al (1987) Prot. Eng. 1 499-505). The lowering of the level of the HAMA response to the chimeric antibodies leads to the expectation that further engineering of the variable region outside of the antigen binding site may abolish the response to these regions and further reduce any adverse response.
A more complex form of engineering of an antibody involves the redesign of the variable region domain so that the amino acids constituting the murine antibody binding site are integrated into the framework of a human antibody variable region. This has led to the reconstitution of full antigen binding activity in a number of cases (Co et al (1990) J. Immunol. 148 1149-1154; Co et al (1992) Proc. Natl. Acad. Sci. USA 88 2869-2873; Carter et al (1992) Proc. Natl. Acad. Sci. 89 4285-4289; Routledge et al (1991) Eur. J. Immunol. 21 2717-2725 and International Patent Specifications Nos. WO 91/09967; WO 91/09968 and WO 92/11383).
Naturally occurring and engineered human antibodies may be regarded as bifunctional agents, with the N-terminal variable region responsible for antigen binding and sequences within the C-terminal part responsible for determining interactions with the various cell types which participate in immune responses. Recognition of these effector sites on antibodies by specific cell surface receptors on cytotoxic cells can result in antibody-dependent cellular cytotoxicity and complement mediated lysis. This can result in killing of the cell presenting the antigen.
E-selectin is expressed on the surface of endothelial cells. The loss of endothelial cells as a result of antibody bound to target antigen is highly undesirable. Endothelial cells make up the endothelium which forms a barrier between the tissues of the body and the vascular system. The loss of or damage to the structural integrity of the endothelium is extremely disadvantageous and can lead to oedema and vasculitis. It is highly advantageous therefore to avoid depletion of the endothelial cell population while blocking the target antigen. Recent papers by Podolsky et al (J. Clin. Invest. 92 (1993) 372-380), Westphal et al Clin. Exp. Immunol. 96 444-449 (1994), and the Editorial Lancet 337 (1991) confirm that the use of whole antibody is undesirable due to undesirable effector functions mediated via the Fc region of the antibody. Another group has attempted to overcome the problem of undesirable effector functions by the use of antibody fragments lacking the effector signals which result in antibody-dependent cellular cytotoxicity (Mulligan et al, J. Clinical Investigation 88, 1396-1406 (1991)). Antibody fragments are known, however, to have a short half-life (Pimm et al Nuclear Medicine Communication 10, 585-593 (1989); Molthoff et al Br. J. Cancer 65, 677-683 (1992) and Buist et al Cancer Res. 53 5413-5418 (1993)) making their therapeutic usefulness in the treatment of many diseases extremely limited.
The present invention provides a novel solution to this problem of preventing depletion of the target endothelial cell population. We have found that by making a whole anti-E-selectin antibody of neutral isotype it is possible to produce a therapeutically useful antibody which does not result in endothelial cell depletion.
In a first aspect the invention therefore provides an antibody having specificity for E-selectin characterised in that said antibody is a whole antibody of neutral isotype.
In a preferred embodiment of the first aspect of the invention the antibody has specificity for human E-selectin.
The antibodies according to the invention preferably recognise the E-selectin lectin or EGF domain.
As used herein the term xe2x80x98wholexe2x80x99 antibody is used to denote an antibody comprising substantially full length heavy and light chains, and antibodies to which amino acids have been substituted, altered, added and/or deleted.
The approach of using a whole antibody of neutral isotype has not been tried before in this area. The term xe2x80x98neutral isotypexe2x80x99 means that the interactions with antibody Fc receptors i.e. FcRI, FcRII and FcRIII and complement are absent or so weak as to cause minimal detrimental physiological effects such as antibody dependent cellular cytotoxicity (ADCC) and/or complement mediated lysis and also the antibody produces a minimal immune response in the host. As used herein the term xe2x80x98minimal immune responsexe2x80x99 is used to denote a typical primate immune response to an iv injection of a human or engineered human antibody.
Anti-E-selectin antibody may be prepared using well-known immunological techniques employing E-selectin as antigen. Any suitable host may, for example, be immunised with E-selectin or activated HUVEC (human umbilical vein endothelial cells) and splenocytes or lymphocytes recovered and immortalised using for example the method of Kohler et al, Eur. J. Immunol. 6, 511 (1976). The resulting cells are diluted and cloned to obtain a single genetic line producing anti-E-selectin antibodies in accordance with conventional practice. Where it is desired to produce recombinant anti-E-selectin antibodies these may be produced using methods well known in the art.
Several regions of the Fc region of antibodies have been implicated in modulating effector functions (see for example European Patent No. 307434B and Lund et al (1991) J. Immunol. 147 2657-2662). For example Lund et al (1991) and other groups have implicated Leu 235 in the CH2 domain of human IgG3 heavy chain in binding of antibody to the high affinity receptor on mononuclear phagocytes (FcRI). Thus by altering this residue it is possible to produce an antibody lacking FcRI binding activity.
In a further aspect the invention provides an antibody characterised in that said antibody is a whole antibody of neutral isotype having specificity for E-selectin wherein one or more amino acid residues in the Fc region of said antibody has been altered from that in the naturally occurring sequence.
In a preferred embodiment of this aspect the invention therefore provides an antibody characterised in that said antibody is a whole antibody of neutral isotype having specificity for E-selectin wherein one or more amino acid residues in the Fc region of said antibody including residue 235 in the CH2 domain has been altered from that in the naturally occurring sequence.
Residue 235 occurs in the N-terminal region of the CH2 domain of the heavy chain. We have found altering the naturally occurring residue e.g. residue Leu 235 to an alanine is particularly advantageous since the conservative nature of the amino acid change is less likely to produce undesirable structural changes in the molecule, which may result in the antibody being immunogenic.
In a preferred embodiment the antibody of the invention has an alanine residue at position 235 in the CH2 domain and in a particularly preferred embodiment the antibody has a human xcex34 isotype.
Alteration of antibody side chain interaction with FcR1 receptor may similarly be achieved by replacing leucine with isoleucine, valine, threonine or glutamic acid. It will be readily apparent to one skilled in the art that a number of other substitutions are possible within the overall aim of not introducing any undesirable structural changes in the molecule, which may lead to immunogenicity.
Where the antibody has isotypes such as human xcex31, xcex32 or xcex33 it will be apparent to one skilled in the art that further alterations to amino acid residues will be required to alter FcRI, FcRII and FcRIII binding where appropriate and also to minimise complement fixation. For example, alteration of residues 235 and 234 of the heavy chain of xcex31, and xcex33 antibodies is known to affect FcRI and FCRII binding (Burton and Woof Adv. Immunol (1992) 1: Academic Press), and similarly amino acid residues 234, 235, 330, 331, 318, 320 and 322 have been shown to be involved in binding and activation of complement (Xu et al (1994) J. Biol. Chem 269 (5) 3469-3474, Published European Patent No. EP 307434B and published International Patent Application No. WO 9429351). See also, Woof et al Mol. Immunol 23 319-330 (1986), Burton et al Nature 288 338-44 (1980); Burton Mol. Immunol 22 161-206 (1988); Leatherbarrow et al Mol. Immunol 22 407-415 (1985); and Duncan et al Nature 332 563-4 (1988). The antibodies of the invention preferably have a human isotype.
The standard techniques of molecular biology may be used to prepare DNA sequences coding for the antibodies according to the invention. Desired DNA sequences may be synthesised completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.
Suitable processes which may be used to alter the residue at position 235 include the PCR strand overlap procedure PCR mutagenesis, as described for example in the teaching of PCR Technology Principles and Applications for DNA Amplification (1989), Ed. H. A. Erlich, Stockton Press, N.Y., London, and oligonucleotide directed mutagenesis (Kramer et al, (Nucleic. Acid. Res. 12, 9441 (1984)). Suitable methods are also disclosed in Published European Patent No. EP307434B.
The alteration at position 235 or any other position of the molecule may be introduced at any convenient stage in the antibody production process. For example, where the antibody is a CDR-grafted antibody, the change may be made before, or more conveniently after CDR-grafting has been completed. This is described in more detail in the accompanying examples.
In a preferred embodiment the antibody molecule of the invention is an IgG and most preferably has a human xcex34 isotype.
It has further been found (Angal et al (1993) Molecular Immunol 30 105-108) that the sequence of the hinge region of antibodies of the xcex34 isotype, i.e. Cys-Pro-Ser-Cys can give rise to alternative forms of the antibody in association with correctly folded and assembled forms. This can be overcome by altering the Ser residue at position 228 to a Pro residue using for example site directed or oligonucleotide directed mutagenesis. The anti-E selectin antibodies of the invention which are of xcex34 isotype preferably have the sequence Cys-Pro-Pro-Cys at the hinge region, and most preferably also have an alanine residue at position 235 in the CH2 domain.
The residue numbering used herein is according to the EU index described in Kabat et al [(1991) in: Sequences of Proteins of Immunological Interest, 5th Edition, United States Department of Health and Human Services].
The antibodies of the invention are preferably engineered human antibodies, most preferably CDR-grafted antibodies.
In a preferred embodiment the invention therefore provides an engineered human antibody having specificity for E-selectin characterised in that said antibody is a whole antibody of neutral isotype.
The term engineered human antibody molecule is used to describe a molecule having an antigen binding site derived from an immunoglobulin from a non-human species, the remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin. The antigen binding site may comprise either complete variable regions fused onto human constant domains or only the complementarity determining regions (CDRs) grafted onto appropriate human framework regions in the variable domains.
The whole anti-E-selectin antibodies of neutral isotype according to the invention are preferably engineered human antibodies wherein one or more amino acid residues in the Fc region of the antibody has been altered from that in the naturally occurring sequence.
The present invention provides an engineered human antibody molecule. having specificity for E-selectin characterised in that said antibody is a whole antibody of neutral isotype and has an antigen binding site wherein at least one of the complementarity determining regions of the variable domain is derived from a non-human monoclonal antibody and the remaining immunoglobulin-derived parts of the engineered human antibody molecule are derived from a human immunoglobulin.
The engineered human antibody molecule may comprise a chimeric antibody or a CDR-grafted antibody. When the engineered human antibody molecule comprises a CDR-grafted antibody, the heavy and/or light chain variable domains may comprise only one or two non-human derived CDRs; though preferably all three heavy and light chain CDRs are derived from the non-human antibody.
The non-human antibody is preferably ENA-2. ENA-2 is a mouse IgG1/kappa antibody that binds to the lectin/EGF region of human E-selectin and blocks cell binding. (Leeuwenberg et al (1990) Transplantation Proceedings 22 (4) 1991-1993).
The human immunoglobulin derived parts of the engineered human antibody molecule may be derived from any suitable human immunoglobulin. For instance where the engineered human antibody molecule is a CDR-grafted antibody molecule, appropriate variable region framework sequences may be used having regard to class/type of the donor antibody from which the antigen binding regions are derived. Preferably the type of human framework used is of the same/similar class/type as the donor antibody. Advantageously the framework is chosen to maximise/optimise homology with the donor antibody sequence particularly at positions spacially close or adjacent to the CDRs. Examples of human frameworks which may be used to construct CDR-grafted antibodies are LAY, POM, TUR, TEI, KOL, NEWM, REI and EU; for instance KOL and NEWM for the heavy chain and REI for the light chain and EU for both the heavy chain and light chain.
In a preferred method the human frameworks are chosen by comparing the sequences of the donor and acceptor heavy and light chains and choosing the human framework sequence which is most homologous to the donor antibody.
The ENA-2 Vh domain shows closest sequence homology to group 1 human heavy chains and consequently the group 1 human antibody Eu was chosen as the framework for both the heavy and light chain variable domains.
The light or heavy chain variable domains of the engineered human antibody molecule may be fused to human light or heavy chain constant domains as appropriate, (the term Fc region and xe2x80x98heavy chain constant domainsxe2x80x99 as used herein are to be understood to include hinge regions unless specified otherwise). The human constant domains of the engineered human antibody molecule, where present, may be selected having regard to the proposed function of the antibody, in particular the lack of effector functions which may be required.
For example, the heavy chain constant domains fused to the heavy chain variable region may be human IgA, IgG or IgM domains. Preferably human IgG domains are used. Depending on the choice of human constant domains it may be necessary to alter specific amino acid residues to remove any undesirable effector function in order to produce an antibody of neutral isotype by, for example, using site directed or oligonucleotide directed mutagenesis. Light chain human constant domains which may be fused to the light chain variable region include human Lambda or human Kappa chains.
Analogues of human constant domains may alternatively be advantageously used. These include those constant domains containing one or more additional amino acids than the corresponding human domain or those constant domains wherein one or more existing amino acids of the corresponding human domain has been substituted, added, deleted or altered. Such domains may be obtained, for example, by oligonucleotide directed mutagenesis.
Also human constant region domains of the engineered human antibody molecule may be selected having regard to the neutral isotype required for the antibody as defined previously.
By appropriate choice of immunoglobulin isotype it is possible to produce an antibody where antibody dependent complement fixation and where interaction with FcRI, FcRII and FcRIII are minimised e.g. by choosing a human xcex34 isotype.
The invention further provides a process for the production of an antibody having specificity for E-selectin characterised in that said antibody is a whole antibody of neutral isotype which process comprises:
a) producing in an expression vector an operon having a DNA sequence which encodes said antibody heavy or light chain;
b) producing in an expression vector an operon having a DNA sequence which encodes a complementary antibody heavy or light chain;
c) transfecting a host cell with both operons and
d) culturing the transfected cell line to produce the antibody.
According to a preferred embodiment of this aspect of the invention there is provided a process for producing an engineered human antibody having specificity for E-selectin characterised in that said antibody is a whole antibody of neutral isotype which process comprises:
a) producing in an expression vector an operon having a DNA sequence which encodes an antibody heavy or light chain comprising a variable domain wherein at least one of the CDRs of the variable domain is derived from a non-human immunoglobulin and the remaining immunoglobulin-derived parts of the antibody chain are derived from a human immunoglobulin;
b) producing in an expression vector an operon having a DNA sequence which encodes a complementary antibody light or heavy chain comprising a variable domain wherein at least one of the CDRs of the variable domain is derived from a non-human immunoglobulin and the remaining immunoglobulin-derived parts of the antibody chain are derived from a human immunoglobulin;
c) transfecting a host cell with both operons; and
d) culturing the transfected cell line to produce the engineered human antibody molecule.
The CDRs of the variable domain are preferably derived from the same non-human immunoglobulin which is most preferably ENA-2.
In a particularly preferred embodiment of this aspect of the invention, at least one of the expression vectors contains a DNA sequence encoding an antibody heavy chain wherein one or more amino acid residues in the Fc region of said antibody most preferably including residue 235 in the CH2 domain has been altered from that in the naturally occurring sequence
The change at amino acid residue 235 or at any other position may also be made after the whole antibody has been assembled using techniques such as site directed mutagenesis.
The cell line may be transfected with two vectors, the first vector containing the operon encoding the light chain-derived polypeptide and the second vector containing the operon encoding the heavy chain derived polypeptide. Preferably the vectors are identical except in so far as the coding sequences and selectable markers are concerned so as to ensure as far as possible that each polypeptide chain is equally expressed.
Alternatively, a single vector may be used, the vector including a selectable marker and the operons encoding both light chain- and heavy chain-derived polypeptides.
In further aspects, the invention also includes DNA sequences coding for the heavy and light chains of the antibodies of the present invention, cloning and expression vectors containing these DNA sequences, host cells transformed with these DNA sequences and processes for producing the heavy or light chains and antibody molecules comprising expressing these DNA sequences in a transformed host cell.
The general methods by which the vectors may be constructed, transfection methods and culture methods are well known per se (see for example Maniatis et al (1982) (Molecular Cloning, Cold Spring Harbor, N.Y.) and Primrose and Old (1980) (Principles of Gene Manipulation, Blackwell, Oxford) and the examples hereinafter).
The DNA sequences which encode the ENA-2 heavy and light chain variable domain amino acid sequences (and the corresponding deduced amino acid sequences) are given hereafter in FIG. 1.
DNA coding for human immunoglobulin sequences may be obtained in any appropriate way. For example, amino acid sequences of preferred human acceptor frameworks such as, LAY, POM, KOL, REI, EU, TUR, TEI and NEWM are widely available to workers in the art.
The standard techniques of molecular biology may be used to prepare DNA sequences coding for CDR-grafted products. Desired DNA sequences may be synthesised completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate. For example, oligonucleotide directed synthesis (Jones et al (1986) Nature 321 522-525). Also oligonucleotide directed mutagenesis of a pre-existing variable domain region (Verhoeyen et al (1988) Science 239 1534-1536; Reichmann et al (1988) Nature 332 323-327).
Enzymatic filling-in of gapped oligonucleotides using T4 DNA polymerase (Queen et al (1989) Proc. Natl. Acad. Sci. USA 86 10029-10033; International Patent Application No. WO 90/07861) may be used.
Any suitable host cell/vector system may be used for expression of the DNA sequences coding the antibody heavy and light chains e.g. for the chimeric or CDR-grafted heavy and light chains. Bacterial e.g. E.coli and other microbial systems may be used. Eucaryotic e.g. mammalian host cell expression systems may also be used to obtain antibodies according to the invention, particularly for production of larger chimeric or CDR-grafted antibody products. Suitable mammalian host cells include CHO cells and myeloma or hybridoma cell lines, for example NSO cells. NSO cells are particularly preferred.
In the engineered human antibody according to the invention, the heavy and light chain variable domains may comprise either the entire variable domains of a non-human antibody such as the murine antibody ENA-2, or may comprise framework regions of a human variable domain having grafted thereon one, some or all of the CDRs of a non-human antibody such as the murine antibody ENA-2. Thus the engineered human antibody may comprise a chimeric engineered human antibody or a CDR-grafted engineered human antibody.
When the engineered human antibody is a CDR-grafted antibody, in addition to the CDRs, specific variable region framework residues may be altered to correspond to non-human e.g. ENA-2 mouse residues. Preferably the CDR-grafted antibodies of the present invention include CDR-grafted antibodies as defined in our International Patent Specification No. WO-A-91/09967. The disclosure of WO-A-91/09967 is incorporated herein by reference.
Preferably the CDRs of the heavy chain correspond to the Kabat defined MAb ENA-2 CDRs at all of CDR1 (31 to 35), CDR2 (50 to 65) and CDR3 (95 to 102). In addition the heavy chain may have mouse ENA-2 residues at one or more of residues 48, 67, 69, 73, 93 and 94. Similarly the light chain may have mouse ENA2 residues at positions 48, 60, 63, 70, 111 and 113.
The present invention also includes therapeutic and diagnostic compositions comprising the antibodies of the invention and the uses of these products and the compositions in therapy and diagnosis. Such compositions typically comprise an antibody according to the invention together with a pharmaceutically acceptable excipient, diluent or carrier, e.g. for in vivo use.
Thus in a further aspect the invention provides a therapeutic or diagnostic composition comprising an antibody according to the invention in combination with a pharmaceutically acceptable excipient diluent or carrier.
The invention also provides a process for the preparation of a therapeutic or diagnostic composition comprising admixing an antibody according to the invention together with a pharmaceutically acceptable excipient, diluent or carrier.
The antibody may be the sole active ingredient in the therapeutic or diagnostic composition or may be accompanied by one or more other active ingredients. The therapeutic and diagnostic compositions may be in unit dosage form, in which case each unit dose comprises an effective amount of the antibody of the invention.
Furthermore, the invention also provides methods of therapy and diagnosis comprising administering an effective amount of an antibody according to the invention to a human or animal subject.
The antibodies and compositions may be for administration in any appropriate form and amount according to the therapy in which they are employed.
The therapeutic or diagnostic composition may take any suitable form for administration, and, preferably is in a form suitable for parenteral administration e.g. by injection of infusion, for example by bolus injection or continuous infusion. It may for example be administered intravenously, intramuscularly, intradermally or intraperitoneally. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents.
Alternatively, the antibody or composition may be in dry form, for reconstitution before use with an appropriate sterile liquid. The antibody may also be formulated for topical administration.
If the antibody or composition is suitable for oral administration, the formulation may contain, in addition to the active ingredient, additives such as: starchxe2x80x94e.g. potato, maize or wheat starch or cellulosexe2x80x94or starch derivatives such as microcrystalline cellulose; silica; various sugars such as lactose; magnesium carbonate and/or calcium phosphate. It is desirable that, if the formulation is for oral administration it will be well tolerated by the patient""s digestive system. To this end, it may be desirable to include in the formulation mucus formers and resins. It may also be desirable to improve tolerance by formulating the antibody or compositions in a capsule which is insoluble in the gastric juices. It may also be preferable to include the antibody or composition in a controlled release formulation.
In a still further aspect of the invention, there is provided a method of treatment of a human or animal subject suffering from or at risk of a disorder associated with increased E-selectin expression the method comprising administering to the subject an effective amount of the antibody or composition of the invention. In particular, the human or animal subject may be suffering from an inflammatory disorder such as a skin disorder e.g. psoriasis.
The antibodies of the invention are particularly useful in the treatment of inflammatory diseases generally. They are particularly useful in the treatment of inflammatory skin diseases e.g. psoriasis, contact dermatitis and eczema; inflammatory bowel disease e.g. Crohn""s disease and ulcerative colitis, in lung inflammatory disorders, e.g. ARDS; arthritis, e.g. rheumatoid arthritis; vasculitis, liver disease e.g. alcoholic hepatitis and cirrhosis, and thermal trauma.
Therapeutic and diagnostic uses typically comprise administering an effective amount of an antibody according to the invention to a human subject. The exact dose to be administered will vary according to the use of the antibody and on the age, sex and condition of the patient but may typically be varied from about 0.1 mg to 1000 mg for example from about 1 mg to 500 mg. The antibody may be administered as a single dose or in a continuous manner over a period of time. Varying doses may be repeated as appropriate. The antibody may be formulated in accordance with conventional practice for administration by any suitable route and may generally be in a liquid form (e.g. a solution of the antibody in a sterile physiologically acceptable buffer) for administration by for example an intravenous, intraperitoneal or intramuscular route.
Since the whole antibodies are of neutral isotype the interaction with Fc receptors will be minimal. This has the effect that such an antibody should block greater than 80% of human neutrophil binding to E-selectin in an in vitro assay and furthermore that this may be observed irrespective of the FcR status of the donor. We believe this may be a considerable advantage in that the antibody is suitable for administration to all patients thereby avoiding the necessity of determining the FcR status of the patient prior to administration of the antibody.
The antibodies according to the invention in in vitro assays show minimal binding to FcR1 carrying cells that do not express E-selectin thereby minimising the potential for ADCC.
The antibody according to the invention may also be used for inflamed site-specific delivery of drugs, nucleic acid and proteins and fragments thereof, radionuclides or chelated metals and other therapeutic agents. The therapeutic agents may be linked directly to the antibody or via a carrier such as for example, a liposome, virus or viral particle where the therapeutic agent to be delivered is incorporated as part of the carrier. This technology is well known in the art. Similarly the antibodies of the invention may also be used diagnostically in the identification of areas of inflammation. The antibodies may be unlabelled or may be labelled for example with a radiolabel, e.g. a radionuclide; a chelated metal; a photochemical reagent, e.g. a fluorescent compound; a dye, or a label detectable via reaction with an enzyme, substrate or cofactor, or a compound detected via NMR or ESR spectroscopy.